Recombinant Schizosaccharomyces pombe Putative cell agglutination protein C188.09c (SPCC188.09c), partial

What are the basic characteristics of the SPCC188.09c gene in Schizosaccharomyces pombe?

SPCC188.09c is a protein-coding gene in Schizosaccharomyces pombe (fission yeast) with Entrez Gene ID 2539201. The gene encodes a predicted cell surface glycoprotein with mRNA sequence NM_001023202.2 and protein sequence NP_588212.1 . The gene was identified and annotated as part of the comprehensive genome sequencing project for S. pombe conducted by Wood et al. and published in Nature .

To characterize this gene in your research, standard molecular biology techniques should be employed, including PCR amplification of the gene sequence from genomic DNA, followed by sequence verification against reference databases. For expression analysis, quantitative PCR (qPCR) with gene-specific primers is recommended, comparing expression levels across different growth conditions and cell cycle stages.

What expression vectors are recommended for recombinant production of SPCC188.09c?

For recombinant expression of SPCC188.09c, several vector systems can be employed depending on your experimental goals. For homologous expression within S. pombe, pREP series vectors offer thiamine-repressible expression control, which allows for regulated protein production. The specific vector selection should be based on the required expression level (pREP1 for high expression, pREP41 for moderate, and pREP81 for low expression).

For experimental protocols, transform S. pombe cells using the lithium acetate method with the following modifications:

• Culture cells to mid-log phase (OD600 0.5-0.7) in appropriate media

• Harvest cells by centrifugation at 3000g for 5 minutes

• Wash with 0.1M lithium acetate

• Resuspend in transformation mixture containing the vector

• Heat shock at 42°C for 15 minutes

• Plate on selective media lacking appropriate nutrients

For heterologous expression in E. coli, commercially available cDNA ORF clones can serve as starting material, with subsequent subcloning into expression vectors containing appropriate tags for purification .

How does S. pombe compare to other yeast models for studying cell surface proteins?

Schizosaccharomyces pombe offers distinct advantages for studying cell surface proteins compared to Saccharomyces cerevisiae. S. pombe is more similar to humans in several aspects of cellular biology, including more complex centromeres, origins of replication, certain histone modifications, and specifics of cell-cycle control . These similarities make S. pombe potentially more relevant for translational research.

When designing experiments to study cell surface proteins in S. pombe versus S. cerevisiae, consider these methodological differences:

• Cell wall composition: S. pombe has primarily α-1,3-glucan and β-1,3-glucan, requiring different enzymatic treatments for spheroplast preparation:

  • For S. pombe: Use Zymolyase-20T supplemented with Novozyme 234

  • For S. cerevisiae: Standard Zymolyase-100T is usually sufficient

• Membrane fractionation: S. pombe requires stronger mechanical disruption:

  • Use glass bead homogenization with 8-10 cycles of 30-second vortexing with intermittent cooling

• Protein expression systems: For S. pombe, thiamine-repressible promoters (nmt1) offer tight regulation, while S. cerevisiae often employs galactose-inducible systems.

What experimental design is optimal for functional characterization of SPCC188.09c?

For comprehensive functional characterization of SPCC188.09c, a multi-pronged approach is recommended, combining gene deletion, controlled expression, and protein localization studies. The experimental workflow should include:

• CRISPR-Cas9 mediated gene deletion:

  • Design guide RNAs targeting the 5' and 3' regions of SPCC188.09c

  • Include homology-directed repair template with selectable marker

  • Verify deletion by PCR and sequencing

• Phenotypic analysis of the deletion mutant:

  • Growth rate measurement in different media and stress conditions

  • Cell morphology assessment through microscopy

  • Cell wall integrity tests using calcofluor white and congo red sensitivity

  • Cell agglutination assays with quantitative measurement

• Protein localization studies:

  • C-terminal tagging with GFP or mCherry via homologous recombination

  • Live cell imaging across different cell cycle stages

  • Co-localization with established cell membrane markers

• Controlled expression studies:

  • Use the nmt1 promoter system with varying strengths (3X repression levels)

  • Analyze dose-dependent phenotypes

  • Measure impact on cell surface properties through atomic force microscopy

When designing these experiments, ensure proper controls are included and perform multiple biological replicates to reduce experimental uncertainty3. Adjust experimental parameters systematically to identify optimal conditions for each assay.

How can recombination studies in S. pombe inform understanding of SPCC188.09c function?

S. pombe presents an excellent model for recombination studies that may elucidate SPCC188.09c function, particularly if this cell surface protein plays a role in cell-cell recognition during mating or meiosis. To investigate this:

• Design a recombination assay using the following approach:

  • Create strains with different selectable markers flanking the SPCC188.09c locus

  • Induce meiosis using nitrogen starvation protocols

  • Measure recombination frequency in wild-type versus SPCC188.09c mutant backgrounds

• Analyze chromosome dynamics during meiosis:

  • Utilize the horsetail movement phase unique to S. pombe meiosis

  • Track telomere movement using fluorescent tags

  • Compare dynamics in wild-type versus mutant cells

Importantly, S. pombe lacks a fully developed synaptonemal complex and crossover interference, allowing for study of essential recombination features without these complexities . The horsetail movement during meiosis requires dynein components (Dhc1, Dlc1), dynactin component (Ssm4), and SPB components (Mcp6, Kms1) . When designing experiments to track this process:

• Use live-cell imaging with fluorescently tagged chromosomal regions

• Measure movement parameters:

  • Velocity of nuclear movement

  • Duration of horsetail phase

  • Pairing efficiency of homologous regions

• Compare results between wild-type and SPCC188.09c mutant cells to determine if this cell surface protein affects cell-cell communication during meiosis.

What data analysis approaches are recommended for interpreting SPCC188.09c localization experiments?

When analyzing localization data for SPCC188.09c protein, implement rigorous quantitative approaches rather than relying solely on qualitative observations. Follow this methodological framework:

• Image acquisition protocol:

  • Capture Z-stack images (minimum 0.3μm intervals)

  • Obtain multiple fields (>10) per condition

  • Include appropriate fluorescent controls for background subtraction

• Quantitative analysis workflow:

  • Perform deconvolution to improve signal-to-noise ratio

  • Apply consistent thresholding across all samples

  • Measure fluorescence intensity distribution across cell compartments

  • Calculate Pearson's correlation coefficient for co-localization studies

• Statistical validation:

  • Analyze minimum 100 cells per condition

  • Perform appropriate statistical tests (ANOVA with post-hoc tests)

  • Plot data using box plots or violin plots rather than bar graphs to show data distribution

When presenting results, create quantitative tables showing:

To reduce experimental uncertainty, perform multiple trials under varied conditions, such as different growth phases and stress conditions, to capture dynamic localization patterns3.

How can researchers address data contradictions when studying SPCC188.09c function?

When confronting contradictory findings regarding SPCC188.09c function, implement a systematic troubleshooting approach:

• Validate strain authenticity:

  • Sequence verify the SPCC188.09c locus in all strains

  • Check for second-site suppressors through whole-genome sequencing

  • Create fresh deletion/tagging constructs with alternative selection markers

• Cross-validate with complementary techniques:

  • If microscopy and biochemical fractionation show different localization patterns, employ orthogonal methods:

    • Immunogold electron microscopy for high-resolution localization

    • Split-GFP complementation assays for protein-protein interactions

    • Mass spectrometry of purified membrane fractions

• Consider condition-dependent functions:

  • Test function under various stress conditions:

    • Osmotic stress (0.4M-1.2M KCl or sorbitol)

    • Temperature stress (20°C, 30°C, 37°C)

    • Cell wall stress (calcofluor white, congo red)

    • Stationary phase versus logarithmic growth

• Create an experimental matrix to systematically test all variables:

When interpreting data, be alert to potential errors in experimental setup. As noted in the AP Physics experimental design guidance, carefully reading and understanding all requirements before designing experiments is crucial to avoid misinterpretation3. Plot data carefully, using appropriate scales, and always include error bars representing statistical uncertainty.

What purification strategies are most effective for SPCC188.09c as a cell surface glycoprotein?

Purifying cell surface glycoproteins like SPCC188.09c requires specialized approaches to maintain native structure and glycosylation patterns. Implement this optimized protocol:

• Membrane preparation:

  • Harvest cells in mid-log phase (OD600 0.5-0.7)

  • Wash with cold buffer containing protease inhibitors

  • Disrupt cells using glass bead homogenization (8-10 cycles, 30 seconds each)

  • Separate membrane fraction via ultracentrifugation (100,000g, 1 hour)

• Detergent solubilization optimization:

  • Test panel of detergents at various concentrations:

    • Mild: Digitonin (0.5-1%), DDM (0.5-1%)

    • Moderate: CHAPS (0.5-2%), Triton X-100 (0.5-1%)

    • Strong: SDS (0.1-0.5%), Sarkosyl (0.5-1%)

  • Solubilize at 4°C for 1-2 hours with gentle rotation

• Affinity purification options:

  • For tagged proteins: Use appropriate affinity resin (Ni-NTA for His-tag)

  • For native protein: Consider lectin affinity (ConA, WGA) targeting glycan moieties

  • Elute using competitive binding or pH/salt gradients

• Further purification and analysis:

  • Size exclusion chromatography to separate oligomeric states

  • Ion exchange chromatography for additional purity

  • Glycosylation analysis using PNGase F or similar enzymes

After purification, verify identity using mass spectrometry and assess purity by SDS-PAGE. For glycosylation analysis, compare migration patterns before and after deglycosylation treatments.

How should researchers design experiments to study SPCC188.09c interactions with other cell surface proteins?

To investigate potential interactions between SPCC188.09c and other cell surface proteins, employ a multi-technique approach:

• In vivo interaction studies:

  • Bimolecular Fluorescence Complementation (BiFC):

    • Fuse SPCC188.09c to N-terminal half of Venus/YFP

    • Fuse candidate interacting proteins to C-terminal half

    • Analyze reconstituted fluorescence using microscopy

  • Proximity-dependent biotin identification (BioID):

    • Fuse SPCC188.09c to a promiscuous biotin ligase (BioID2)

    • Purify biotinylated proteins using streptavidin

    • Identify by mass spectrometry

• In vitro binding assays:

  • Surface Plasmon Resonance (SPR):

    • Immobilize purified SPCC188.09c on sensor chip

    • Flow potential binding partners at varying concentrations

    • Determine binding kinetics (kon, koff, KD)

  • Pull-down assays:

    • Express GST-tagged SPCC188.09c

    • Incubate with cell lysates or purified candidate proteins

    • Identify binding partners by immunoblotting or mass spectrometry

When designing these experiments, create appropriate controls:

• Use unrelated membrane protein as negative control

• Include known interacting protein pairs as positive control

• Generate predicted non-binding mutants of SPCC188.09c

Present interaction data in a clear table format:

To reduce experimental uncertainty, perform multiple trials and vary experimental conditions systematically3.

How does SPCC188.09c compare structurally and functionally to homologous proteins in other yeasts?

To conduct a comprehensive comparative analysis of SPCC188.09c with homologs in other yeasts, implement the following methodological approach:

• Sequence-based analysis:

  • Perform BLAST and HMMer searches against fungal proteomes

  • Generate multiple sequence alignments using MUSCLE or MAFFT

  • Construct phylogenetic trees using maximum likelihood methods

• Structural prediction and comparison:

  • Generate 3D structure predictions using AlphaFold or RoseTTAFold

  • Compare predicted structures using DALI or TM-align

  • Identify conserved structural motifs and functional domains

• Experimental validation:

  • Test functional complementation by expressing homologs in S. pombe

  • Analyze localization patterns of homologs when expressed in S. pombe

  • Compare phenotypes of corresponding gene deletions across species

While S. pombe and S. cerevisiae are both yeasts, they are evolutionarily distant, with many proteins showing divergent functions despite sequence similarity . This comparison is particularly valuable as S. pombe aspects are often more similar to humans than those of S. cerevisiae, including more complex centromeres, origins of replication, and certain histone modifications .

Create a comparative table highlighting key differences:

What experimental approaches can determine if SPCC188.09c is involved in meiotic recombination?

To investigate potential roles of SPCC188.09c in meiotic recombination, design experiments that leverage S. pombe's strengths as a model for recombination studies:

• Recombination frequency analysis:

  • Create strains with genetic markers flanking potential recombination sites

  • Induce meiosis by nitrogen starvation

  • Measure recombination frequency in wild-type vs. SPCC188.09c deletion strains

  • Analyze at least 1000 tetrads per condition for statistical significance

• Cytological analysis of recombination intermediates:

  • Visualize Rad51 foci formation by immunofluorescence

  • Track double-strand break (DSB) formation using Southern blotting

  • Monitor crossover formation using physical assays

• Protein interactions during meiosis:

  • Perform co-immunoprecipitation with known recombination factors

  • Use ChIP-seq to identify potential binding sites on chromatin

  • Analyze timing of SPCC188.09c recruitment to meiotic chromosomes

S. pombe offers special advantages for these studies because even the strongest meiotic recombination-deficient mutants produce viable spores at rates of 10-25% compared to wild-type . This occurs because S. pombe has only three chromosomes and possesses a mechanism for actively segregating non-recombinant chromosomes at MI .

For experimental design, follow these methodological principles:

• Include appropriate positive controls (e.g., rec12 deletion for reduced recombination)

• Design experiments with sufficient statistical power (minimum 3 biological replicates)

• Use multiple independent SPCC188.09c mutant strains to confirm phenotypes

What emerging technologies could enhance studies of SPCC188.09c function?

Several cutting-edge technologies offer promising approaches for deeper investigation of SPCC188.09c function:

• Proximity proteomics approaches:

  • TurboID-based proximity labeling in living cells

  • Implementation protocol:

    • Fuse TurboID to SPCC188.09c

    • Express in S. pombe under native or regulated promoter

    • Add biotin for short pulses (10 minutes)

    • Purify biotinylated proteins and identify by mass spectrometry

• Single-cell technologies:

  • Single-cell RNA-seq to identify transcriptional changes in SPCC188.09c mutants

  • Single-cell proteomics to analyze protein expression variability

  • Methodology:

    • Prepare protoplasts using optimized enzymatic digestion

    • Sort single cells using microfluidic devices

    • Process using appropriate single-cell platform

• Cryo-electron tomography:

  • Visualize native cell wall and membrane architecture

  • Compare wild-type and SPCC188.09c mutant cells

  • Technical approach:

    • Prepare cells by high-pressure freezing

    • Acquire tomograms of cell periphery

    • Perform subtomogram averaging of repetitive structures

• Genome-wide interaction screens:

  • CRISPR interference (CRISPRi) screens in SPCC188.09c background

  • Synthetic genetic array analysis with SPCC188.09c as query

When implementing these technologies, design experiments carefully to reduce experimental uncertainty through multiple trials and systematic variation of experimental parameters3.